Protein ubiquitination is an important regulator of cytokine-mediated signal transduction pathways and hematopoietic cell growth. Protein ubiquitination is controlled by the coordinate action of ubiquitin conjugating enzymes and deubiquitinating enzymes. During the last 10 years of this grant, our laboratory has examined the regulation of cytokine-mediated signal transduction events by sequential phosphorylation and ubiquitination of intracellular signaling proteins. Recently, my laboratory has made the surprising observation that another hematopoietic signaling pathway, the Fanconi Anemia (FA) Pathway, is also regulated by highly coordinated monoubiquitination and deubiquitination events. The six cloned FA genes (A, C, D2, E, F, and G) interact in a novel pathway. Five of the FA proteins (A, C, E, F, and G) assemble in a nuclear complex which is required for the monoubiquitination of the downstream FA protein, FANCD2 (Garcia-Higuera et al, Mol Cell, 2001). Disruption of this pathway leads to the characteristic clinical phenotype observed in FA, including developmental abnormalities, progressive bone marrow failure, and predisposition to acute myeloid leukemia. During the next five of years of support, our laboratory plans to (1) devise and employ an in vitro ubiquitination assay to determine the precise requirements for the monoubiquitination of the FANCD2 protein (2) analyze the enzyme(s) involved in the regulated deubiquitination of FANCD2 which occurs during the G2/M transition of the cell cycle or following repair of DNA damage and (3) examine the binding partners of the activated (monoubiquitinated) isoform of FANCD2. In the course of these experiments, we hope to determine novel protein regulators of in vivo hematopoietic stem cell growth and differentiation.